Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry, excessive apoptosis results in cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders, while insufficient apoptosis contributes to the development of cancer, autoimmune disorders and viral infections (Thompson, Science, 1995, 267, 1456-1462).
Although several stimuli can induce apoptosis, little is known about the intermediate signaling events, including inhibition, that connect the apoptotic signal to a common cell death pathway conserved across many species. Recently, major advances have been made in understanding the signaling pathways mediated by the tumor necrosis factor receptor (TNFR) family which signals apoptosis. Two cell surface cytokine receptors of the TNFR family, TNFR-1 and CD95 (Fas/APO-1), act as death receptors and a number of binding proteins have been identified which mediate apoptosis through these receptors (Baker and Reddy, Oncogene, 1998, 17, 3261-3270).
Daxx (also known as Fas binding protein, CENP-C binding protein, dap6 for death associated protein 6 and EAP for Ets-1 associated protein) is a novel signaling protein which interacts with the cytoplasmic domain of Fas/APO-1 acting as a downstream effector in the process of apoptosis (Yang et al., Cell, 1997, 89, 1067-1076). The nucleic acid and protein sequences for human daxx are disclosed in the PCT publication WO 98/34946. Also disclosed are methods for decreasing Jun N-terminal kinase (JNK) signaling pathways by contacting cells with an inhibitor of daxx, wherein the inhibitors are antisense nucleic acids (Yang et al., 1998). Overexpression of daxx enhances Fas-mediated apoptosis and activates the JNK pathway (Chang et al., Science, 1998, 281, 1860-1863; Yang et al., Cell, 1997, 89, 1067-1076). This signaling pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Moriguchi et al., Adv. Pharmacol., 1996, 36, 121-137).
Daxx is widely expressed in human tissues and cloning of the human gene revealed its localization to chromosome 6p21, a genomic region that includes the major histocompatibility complex (MHC) and which is implicated in the pathway for deletion of autoreactive lymphocytes (Herberg et al., J. Mol. Biol., 1998, 277, 839-857; Kiriakidou et al., DNA Cell. Biol., 1997, 16, 1289-1298). This may suggest a role for daxx in autoimmune diseases.
Daxx also interacts with a centromeric protein known as CENP-C. CENP-C is crucial to proper chromosome segregation and mitotic progression. Pluta et al. have demonstrated that daxx colocalizes with CENP-C at interphase-specific centromeres in human cell nucleii and suggest that daxx may play a role in regulating cellular responses to apoptotic stimuli (Pluta et al., J. Cell. Sci., 1998, 111, 2029-2041).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of daxx, and there remains a long felt need for agents capable of effectively inhibiting daxx function.
To date, investigative strategies aimed at modulating daxx function have involved the use of antibodies, molecules that block upstream entities, and gene knock-outs in mice.
Knockout of the daxx gene in mice resulted in extensive apoptosis and embryonic lethality. This was in contrast to the expected result from the loss of a pro-apoptotic gene, that being a hyperproliferative disorder. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo (Michaelson et al., Genes Dev., 1999, 13, 1918-1923).
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of daxx expression.
The pharmacological modulation of daxx activity and/or function may therefore be an appropriate point of therapeutic intervention in pathological conditions and the present invention provides compositions and methods for modulating daxx expression.